The book [18] provides a review of problems and of techniques for administering and assimilating ophthalmic solutions.
Corneal cross-linking (C3) with riboflavin (vitamin B2), shortly called riboflavin-C3, is an innovative technique for treating patients affected by keratoconus and corneal ectasia and consists in administering riboflavin and ultra-violet irradiation (UV-A) for strengthening the corneal tissue [1] [2].
The cross-linking treatment is relatively simple: riboflavin in instilled in the eye and the cornea is irradiated with a properly dosed amount of UV-A rays for five minutes; the procedure is then repeated six times in succession for a total exposition to UV-A rays of 30 minutes.
The most important clinical parameter that should be taken into consideration for establishing the suitability to the cross-linking treatment is the corneal thickness, that must be not smaller than 400 microns.
The objective of this conservative treatment of keratoconus is to delay or hopefully to eliminate the need of corneal transplantation and to improve visual performances of patients by enhancing they quality of life [6] [7].
The cross-linking technique has been used for the treatment of keratoconus, a pathology characterized by a progressive weakening of the cornea for an anomalous laxity of corneal parenchyma due to a reduced cohesion of collagen lamellae of which it is composed. By using UV-A rays and riboflavin, new links among adjacent corneal collagen molecules are created and the treated corneas are thicker and stiffer [3]. Corneas have numerous layers of collagen fibers in the thickness of the parenchyma; the transversal links, the so-called “cross-links” that tie the various layers of collagen among them, contribute in a determinant fashion to the corneal stiffness. The objective of the corneal cross-linking treatment is to increase the degree of rigidity of the corneal tissue through the generation of a greater number of these transversal links.
Topical application of riboflavin on the disepithelized cornea with penetration of about 200 μm and irradiation of riboflavin molecules by UV-A determine the loss of chemical equilibrium of riboflavin molecules with the consequent generation of free radicals. Riboflavin molecules become unstable and stabilize themselves by linking with two collagen fibrils. A series of biochemical “bridges” are formed among collagen fibrils (i.e. cross-linkings) such to produce a general strengthening of the cornea [3].
Actually, the treatment is carried out after having removed the outermost layer of the corneal (i.e. the corneal epithelium). This way of executing the cross-linking treatment (C3-R) of keratoconus and of corneal ectasiae contemplates the preliminary removal of corneal epithelium for favoring penetration of the standard solution of riboflavin-dextran 0.1% (for example the solution marketed by SOOFT ITALIA S.r.l. under the trademark RICROLIN™) in the underlying stroma and the treatment has been standardized under these conditions. According to supporters of this technique, the removal of the epithelial layer would be necessary for ensuring the best possible absorption of the riboflavin solution inside the corneal stroma and thus the maximum effectiveness of the therapy.
Unfortunately, the removal of the corneal epithelium may cause ocular itches or burn the day after the treatment and in the immediately successive days and transient blurring; these symptoms are well known and persist until the corneal epithelium has not been restored and are usually treated, in the successive days [4-7] after the C3-R, with non-steroidal anti-inflammatory (NSAID) eye drops, with eye drops based on tear substitutes and analgesics, and with the application of a therapeutic contact lens on the cornea.
Several authors sustain that it could be possible to execute the C3-R treatment by applying the standard methodology without preliminary removal of corneal epithelium, and that such a treatment would be effective and safe as demonstrated by the observed clinical data. According to this technique, the treatment should be executed without preliminarily removing the corneal epithelium (disepithelization). The objective is to avoid that patients experience diseases due to the epithelium removal, intrinsic of the first method, to execute the treatment in an ambulatory and in particular to avoid any risk of post-surgical infections intrinsic in the treatment that contemplates the epithelium removal with a consequent exposition of underlying layers of the cornea. Supporters of this way of executing the treatment suggested to apply riboflavin on the eye for a longer time interval for allowing a better absorption of riboflavin in the stroma before irradiating with UV-A rays [8].
As far as the removal or not of the epithelium is concerned in the treatment with cross-linking of keratoconus and of corneal ectasiae in general, contrasting opinions are reported in literature. The C3-R treatment has been studied and implemented after having removed the corneal epithelium for favoring the penetration of riboflavin in the stroma. To the best knowledge of the authors, no study is available in literature for determining whether or not and how much riboflavin penetrates in corneal stroma [9] by removing or without removing the corneal epithelium.
The execution of cross-linking without preliminary disepithelization has been criticized by numerous authors, who sustain that in this way riboflavin would not pass through the epithelium and that it has not yet been demonstrated whether or not and how much standard solution of riboflavin-dextran 0.1% effectively penetrates in the corneal stroma without removing the epithelium and whether or not the treatment by UV-A rays in a trans-epithelial fashion result equally effective as that carried out after removal of corneal epithelium.
In an attempt to provide effective substances for treating keratoconus with cross-linking technique without removing the corneal epithelium, Dr. Spörl suggested [17] to use benzalkonium chloride for increasing permeability of epithelium and Dr. Pinelli suggested to use tensioactives mixed with riboflavin.
The Italian patent application No. MI2007A002162 [16] discloses a novel solution for the treatment of keratoconus with a trans-epithelial cross-linking technique containing riboflavin and benzalkonium chloride.
Experiments executed by the applicants on human corneas, the results of which are illustrated hereinafter, lead to the conclusion that the second technique carried out using the standard solution or the composition proposed by Dr. Pinelli [16] would not overcome the problems due to the removal of corneal epithelium because it would be destroyed, leaving exposed the underlying layers with consequent risks of infections and alterations of repairing mechanisms. It would be desirable to have a composition containing riboflavin for executing the corneal cross-linking that be capable to cross the corneal epithelium in relatively short times and that do not damage the corneal epithelium, that is the cause of annoying post-surgical symptoms.